The present invention relates to conjugate vaccines for prevention of bacterial infections. More specifically, the invention relates to a conjugate vaccine for nontypeable Haemophilus influenzae comprising lipooligosaccharide from which esterified fatty acids have been removed linked to an immunogenic carrier.
Nontypeable Haemophilus influenzae (NTHi) is a major causative agent for acute otitis media (middle ear infections) and respiratory infections. Acute otitis media and otitis media with effusion are common childhood diseases, second in frequency of occurrence only to the common cold (Stool et al., Pediatr. Infect. Dis. Suppl., 8:S11-S14, 1989). The annual cost of the medical and surgical treatment of otitis media in the United States is estimated at between three and four billion dollars (Berman, New Engl. J. Med., 332:1560-1565, 1995). Moreover, inappropriate antibiotic treatment of otitis media can lead to the emergence of multidrug-resistant bacterial strains. There is currently no vaccine available for prevention of NTHi infection.
Current efforts in developing an NTHi vaccine are focused on cell surface antigens such as outer membrane proteins and pili or fimbria (Kyd et al., Infect. Immun., 63:2931-2940, 1995; Deich et al., Vaccine Res., 2:31-39, 1995). Among these, the most promising is P6 protein which appears to be antigenically conserved and elicits the production of antibodies that are bactericidal in vitro. Lipooligosaccharide (LOS) is a major NTHi cell surface antigen. LOS contains both lipid A and oligosaccharide (OS) components. Because the lipid A component of LOS is toxic, it must be detoxified prior to conjugation to an immunogenic carrier.
Barenkamp et al. (Pediatr. Infect. Dis. J., 9:333-339, 1990) demonstrated that LOS stimulated the production of bactericidal antibodies directed against NTHi. McGehee et al. (Am. Journal Respir. Cell Biol., 1:201-210, 1989) showed that passive immunization of mice with monoclonal antibodies directed against LOS from NTHi enhanced the pulmonary clearance of NTHi.
Green et al. (Vaccines, 125-129, 1994) disclose an NTHi vaccine comprising a conjugate of NTHi OS and the mutant nontoxic diphtheria protein CRM197. The lipid A moiety was removed from LOS by treatment with acid, followed by derivatizing the resulting OS with adipic acid dihydrazide (ADH) and coupling to CRM197. Despite the showing of Barenkamp et al. that LOS stimulated production of bactericidal antibodies against NTHi, the conjugates of Green et al. were determined to be poorly immunogenic after injection into mice. Moreover, the conjugates did not elicit bactericidal antibodies against NTHi.
Thus, there is a need for a vaccine effective against NTHi. The present invention satisfies this need.
One embodiment of the present invention is a conjugate vaccine for nontypeable Haemophilus influenzae (NTHi), comprising NTHi lipooligosaccharide from which esterified fatty acids have been removed (dLOS), and an immunogenic carrier covalently linked thereto. In another aspect of the present invention, the immunogenic carrier is a protein. Preferably, the protein is tetanus toxin/toxoid, NTHi high molecular weight protein, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 protein, or respiratory syncytial virus F and G protein. Most preferably, the protein is tetanus toxoid or NTHi high molecular weight protein.
The present invention also provides a conjugate vaccine for nontypeable Haemophilus influenzae (NTHi), comprising NTHi lipooligosaccharide from which esterified fatty acids have been removed (dLOS), and an immunogenic carrier covalently linked thereto via a linker. Preferably, the linker is adipic acid dihydrazide, xcex5-aminohexanol acid, chlorohexanol dimethyl acetal, D-glucuronolactone or p-nitrophenylamine; most preferably, the linker is adipic acid dihydrazide.
Another embodiment of the invention is isolated NTHi lipooligosaccharide detoxified by removal of ester-linked fatty acids therefrom.
The present invention also provides a pharmaceutical composition comprising the vaccine conjugates described above in a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise an adjuvant. Preferably, the adjuvant is alum.
Another embodiment of the invention is a method of preventing otitis media caused by NTHi in a mammal, comprising administering to the mammal an effective immunoprotective amount of the vaccine described above. Preferably, the mammal is a human. The route of administration may be intramuscular, subcutaneous, intraperitoneal, intraarterial, intravenous or intranasal; most preferably, the administering step is intramuscular. According to another aspect of this preferred embodiment,. the effective dose is between about 10 xcexcg and about 50 xcexcg. The method may further comprise injecting between about 10 xcexcg and about 25 xcexcg at about 2 months and at about 13 months after the administering step. Alternatively, the method may further comprise injecting between about 10 xcexcg and about 25 xcexcg at about 2, 4 and 16 months after the administering step.
According to another aspect of the invention, there is provided a method of detoxifying lipooligosaccharide from NTHi, comprising removing ester-linked fatty acids therefrom. Preferably, the ester-linked fatty acids are removed by treating the LOS with hydrazine.
Still another aspect of the present invention is a method of making a conjugate vaccine against NTHi, comprising:
removing ester-linked fatty acids from NTHi lipooligosaccharide to produce dLOS; and
covalently binding said dLOS to an immunogenic carrier.
Advantageously, the removing step comprises treatment with hydrazine. The method may further comprise the step of attaching dLOS to a linker and attaching the linker to the carrier. Preferably, the linker is adipic acid dihydrazide, xcex5-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone or p-nitrophenylethyl amine;
most preferably, the linker is adipic acid dihydrazide.